As reported in the literatures at home and abroad, there is a kind of proteinase associated with blood coagulation in the majority of the snake venoms from Crotalinae, it is generally called “thrombin-like enzyme” (TLC). The thrombin-like enzyme and thrombin have similar functions, and are both capable of converting fibrinogen in blood plasma into fibrin so as to “coagulate”. So far, it has been found that more than 30 kinds of snake venoms comprise the thrombin-like enzymes, and over 20 kinds of them have been isolated and purified, wherein the full length or part of the amino acid sequences of over 10 kinds of thrombin-like enzymes have been identified. The discovered TLCs generally have a molecular weight ranging from 29 to 45 kDa and most of them are acid glycoproteins.
The primary structures of the protein in the previous found TLC of snake venom are generally in a single-stranded form. The typical TLC product, Reptilase, is a thrombin isolated from the snake venom of Bothrops atrox; the precursor of this thrombin consists of 255 amino acids with a N-terminal leader peptide consisting of 24 amino acids and an active enzyme consisting of 231 amino acids, which is a single-stranded glycoprotein having a relative molecular weight of 39-43 kDa.
With the recently 10 years research work, it was found that TLC of Crotalinae snake venoms may also have a double-stranded structure, linked by disulfide bond. In 1999, a “thrombin-like enzyme” isolated from Agkistrodon acutus was reported by Xin Cheng etc. (University of Science and Technology of China), which was designated as “Agkisacutacin”; the protein consists of two peptide chains, wherein the alpha subunit has a molecular weight of 15 kDa, and the beta subunit has a molecular weight of 14 kDa. Agkisacutacin is able to hydrolyze the alpha chain of fibrinogen. In 2004, two kinds of “thrombin-like enzymes” were isolated from Agkistrodon acutus by Xiao Changhua (Kunming Institute of Zoology, Chinese Academy of Sciences), and the molecular structures of them are both two-stranded structures. The subunit A of TLC I consists of 132 amino acids having a molecular weight of 16 kDa, and the subunit B consists of 123 amino acids having a molecular weight of 14 kDa, and the enzymatic specific activity (ESA) of the TLC I is 160 u/mg. The subunit A of TLC II consists of 122 amino acids having a molecular weight of 15 kDa and subunit B consists of 120 amino acids having a molecular weight of 13 kDa, and the enzymatic specific activity (ESA) of the TLC II is 70 u/mg. The pharmacological experiment shows that both of the two TLC have the function of hemostasis.
The People's Republic of China is rich in plentiful research resources of snake venom. The present inventor isolated a hemocoagulase with high activity from the Chinese Agkistrodon acutus. 